The homeodomain is a small structural motif in proteins that binds to specific target DNA sequences and thereby regulates gene transcription in eukaryotes. The homeodomain amino acid sequence determines the binding affinity and specificity for DNA. Thermal unfolding and temperature-induced changes in the secondary structure of the mutant H52R/NK-2 homeodomain protein (9713 MW) were investigated by differential scanning calorimetry (DSC) and far-UV circular dichroism (CD). In 50 mM Na-phosphate (pH 7.4) buffer, the H52R/NK-2 protein reversibly unfolded with Tm = 56 deg C and an enthalpy change (Delta H) of ca 40 kcal/mol. The unfolding was less cooperative in 50 mM Hepes (pH 7.4) buffer with Tm = 39 deg C and [Delta H] ca 34 kcal/mol. Adding 100 mM NaCl to buffers improved refolding during heating and cooling cycles, as measured by CD. Good agreement was obtained between thermodynamic parameters obtained by DSC and temperature-induced CD changes at 222 nm. However, DSC data for the thermal unfolding of the H52R/NK-2 protein were fitted best to a non-two-state model, with ratios of calorimetric to van't Hoff enthalpy changes less than unity. The specific stabilizing effect of the phosphate anion on the N52R/NK-2 homeodomain mutant protein may relate to DNA binding properties. This and other aspects of the conformational stability of different NK-2 homeodomain mutant proteins currently are being studied.